Method for the repeated transfer of liquids

ABSTRACT

A method for the repeated transfer of liquids from one of several storage containers into a transfer container wherein protective tips are placed onto a tip of a pipette in order to avoid contamination.

This application is a divisional of U.S. Pat. application Ser. No.08/489,580, filed Jun. 12, 1995, now U.S. Pat. No. 5,821,436.

Subject matter of the invention is a method for the repeated transfer ofliquids, a method for the qualitative and quantitative determination ofan analyte in a liquid, and a device that is particularly advantageousto implement this method.

Methods for analyzing body fluids in order to diagnose diseases havebeen known as useful tools for some time now in clinical and medicaldiagnostics. Recently, the analysis of nucleic acids has also been usedas a diagnostic tool. But neither the recently developed decoding of thegenetic code of many organisms nor the use of amplification reactionshas led to a more common use of the analysis of nucleic acids as a test.Amplification reactions also allow the detection of minute amounts ofnucleic acids. When amplifications are carried out according to the PCRprinciple (e.g. U.S. Pat. No. 4,683,195), the samples must be preparedin a particularly careful manner in order to isolate the nucleic acidsas there are many biological substances which may interfere with thefunction of the polymerase used in this method. Due to the extremelyhigh sensitivity of this method, it is also necessary that carry-over beavoided in this procedure under all circumstances.

EP-A-0 389 063 describes a method for amplifying nucleic acids where thebinding of nucleic acids to glass particles in the presence ofguanidinium thiocyanate-containing buffers is mentioned as particularlyefficient. Moreover, DE-A-41 39 664, DE-A-41 27 276, and WO 93/11221describe the use of glass fleeces as a separating material where thecentrifugation steps involved require, however, special devices. Thesecurrently used plastic parts used in the above methods obey to theso-called spin-column method which makes use of centrifugal forces topress the solution through the glass fleece. In this method, a greatvariation of parts is used as so-called spin-column tubes.

EP-A-0 588 564 describes pipette tips which, at their lower more narrowend, have a membrane to which analytes can be immobilized. Thisembodiment is particularly susceptible to a carry-over of liquids fromone container to another as these liquids can usually not be completelyremoved from these membranes. When the pipette tip is then brought intocontact with a membrane, liquids from another container may easily betransferred into this container.

It is the object of the present invention to provide methods and devicesto avoid carry-over of reagents from one test to another. Subject matterof the invention is a method for the repeated transfer of liquids fromone or several storage containers into a transfer container,characterized by die following steps:

a) first transfer of a first liquid into a transfer container through afirst opening of the transfer container,

b) attaching a protective tip onto the first opening of the transfercontainer, said protective tip having an opening, and

c) second transfer of a second liquid into the transfer containerthrough the opening of the transfer container and the protective tip.

Another subject matter of the invention is a method for the qualitativeand quantitative determination of an analyte which makes use of thismethod. The fundamental idea underlying this invention is to reduce thecarry-over of sample liquids to containers from which reagent solutionis taken to be transferred into the sample solution. It is thus possibleto reduce contamination when solutions are transferred from reagentbottles. This idea can be applied to any liquid.

Methods for the repeated transfer of liquids from one or severalcontainers are characterized in that certain quantities of a liquid arerepeatedly taken from the same container or that quantities of differentliquids are repeatedly taken from different containers to be transferredinto a transfer container. The containers from which the liquid is takenmay be any desired container, e.g. storage container, tubes, or bottles.Sample storage containers are containers which hold a sample liquid,e.g. primary cups or tubes. Reagent storage containers hold liquidswhich contain components necessary to carry out a chemical reaction,e.g. a detection reaction. In a particularly preferred manner, thesecontainers contain quantities allowing more than 100 reactions to becarried out.

Sample liquids are those liquids which contain an analyte to bedetermined, e.g. a nucleic acid, a cell, an antigen, an antibody, or thelike. Suitable sample liquids are, hence, body fluids, such as blood orurine, or liquids derived therefrom by adding further components orremoving certain components, e.g. serum or plasma.

The transfer container into which liquids are transferred preferably hastwo openings: a first opening through which the liquid is transferredinto the transfer container, and a second opening through which the gasor liquid is removed from the transfer container. The first opening ofthe transfer container has a preferred cross section of 0.1 to 5 mm² andis preferably smaller than the second opening. The second opening has apreferred cross section between 3 and 20 mm² and preferably has the formof a defined, standardized hub flange. This hub flange serves to connectthe transfer container to an instrument which can be used to removeliquids, e.g. a manual pipetting aide or an automated pipettor. Suchinstruments to handle liquid quantities are known to the expert, e.g.pipetting instruments manufactured by Tecan. In this case, the transfercontainer preferably has an upper opening matching the pipetting arm ofthe Tecan instrument. Owing to the shape of the opening, an essentiallyconical form of the inside and also of the outside of the container arepreferred. With respect to the liquids to be transferred and, ifnecessary, the reactions carried out with them, the wall of the transfercontainers are preferably made of an inert plastic material, e.g.polypropylene, polyethylene, polycarbonate, polyurethane. It ispreferred that the interior formed by the inner wall of the container benot capillary. However, in its interior, the transfer container maycontain agents necessary for carrying out a reaction. These agents mayinclude chemical reagents, but also materials for the immobilization ofindividual components of the first liquid, particularly components ofthe liquid to be detected. Materials for immobilization include tissuesmade of polyester, polyamide, polycarbonate, cellulose, nitrocellulose,or glass. These materials can, however, also have another outer shape,e.g. beads or fleeces. If nucleic acids are to be immobilized to thesematerials, the use of glass fleeces is preferred. If other purposes aredesired, it is also possible to use materials that are conventionallyused in affinity chromatography procedures.

Moreover, the transfer container may feature modifications necessary tofix reagents or immobilization material. If nucleic acids are to beimmobilized to a glass fleece, said glass fleece is preferably fixedwith the aid of an inert plastic net in direction towards the twoopenings of the transfer container. The agents present in the transfercontainer are preferably located at such a distance away from the firstopening that contact with the liquids is not made directly uponcontacting the opening. It is particularly preferred that these agentsbe not attached from the outside to the opening of the transfercontainer.

In a preferred case, the transfer container has the form of aconventional pipette tip. This ensures that the method of the inventioncan be adapted to pipetting instruments. However, the transfer containeralso has an area where a protective tip can be attached. This can bedone in the same way as the transfer container is attached to thepipetting arm. For this purpose, the outside of the container is alsoconfigured to be conical to match the hub flange of the protective tip.The attachment of the protective tip at the transfer container can befurther improved by providing a snap in connection.

FIG. 1 is a conceptual illustration of a sequence of method stepsaccording to the present invention.

FIG. 2 is a diagrammatic representation of a pipetting instrument foruse according to the present invention.

An essential part of the present invention is the use of a protectivetip which prevents the outer surface of the transfer container to comeinto direct contact with the second liquid. This is important in so farthat remains of the first liquid may still adhere to the outer surfaceof the transfer container. These remains could then be transferred intothe second liquid and be a source of contamination. Like the transfercontainer, the protective tip preferably also has two openings of whichthe first serves to transfer the second liquid from a storage container.The second opening serves as a connection to the transfer container. Ina preferred manner, a part of the transfer container which is providedwith the first opening projects into the second opening of theprotective tip. It is preferred that the protective tip tightly restagainst the outer wall of the transfer container to avoid loss ofpressure or liquid and to further reduce the risk of contamination. Atits end, the protective tip, therefore, has a defined standardized formwhich matches the outer form of the transfer container in the area ofthe first opening. Possible material for the protective tip isparticularly the one that is also used for the transfer container. In apreferred manner, the protective tip is an inexpensive disposableplastic product. The protective tip can, hence, have a form similar tothe one of conventional pipette tips. Its interior can, however, be muchsmaller than the commonly used pipette tips as the quantity of liquidtaken up must not be contained in it. In its interior, the protectivetip may contain agents for carrying out a reaction as does the transfercontainer. However, preferred pipette tips are those which do notcontain any such agents.

A typical application of a method where the repeated transfer of liquidsfrom storage container occurs are methods for determining analytes in asample liquid. In this particular case, contamination is particularlycritical as it may falsify the results. Another subject matter of theinvention is, hence, a method for the qualitative and quantitativedetermination of an analyte comprising the following steps:

a) Receiving a sample liquid in which the analyte to be determined iscontained through an opening (11) provided in a first transfer container(10)

b) Immobilizing the analyte in the first transfer container (10)

c) Removing the liquid from the transfer container (10)

d) Attaching a protective tip (20) with an opening (21) to the firstopening (11) of the transfer container (10)

e) Transferring a second liquid into the transfer container (10) throughopenings (21, 11).

The following is a description of such an isolation procedure as knownfrom the isolation of nucleic acids. In the description, reference ismade to FIG. 1. In a first step, a given volume of a sample liquid istransferred into the transfer container in any desired way. This can beaccomplished, for example, by applying a low pressure to the firstopening of the transfer container via the second opening of the transfercontainer (e.g. applying suction by means of a pipetting aid orpipetting instrument, such as manufactured by Tecan, Hamilton orBeckmann). It is possible to apply this low pressure several times todraw in and eject liquid. The opening (11) of the transfer containerextends into the sample liquid in the storage container. The transfercontainer has in its interior a glass fleece that is attached by meansof inert plastic nets and spreads over the entire cross section of thetransfer container. The sample liquid contains the nucleic acid to beisolated, preferably in a buffer to facilitate immobilization of thenucleic acid to the glass fleece. A preferred buffer is GuSCN. The glassfleece used is a WF 264, WF 265 fleece (manufactured by Whatman). Afterincubation for 1 sec to 30 min, preferably 2-10 sec, the liquid isejected from the transfer container into the sample storage container ora waste container. The nucleic acids remain immobilized in the transfercontainer.

A protective tip is then placed onto the lower tip of the transfercontainer. These protective tips may be stored in a storage container,e.g. on the pipetting instrument or be automatically mounted in aposition selected by the instrument. The lower part of the transfercontainer which could come into contact with the second liquid is thusprotected.

Subsequently, the transfer container can be forwarded into a positionwhere the opening of the protective tip extends into a washing liquid.By absorbing washing liquid from the liquid storage container, remainingsample liquid which may still adhere to the immobilized nucleic acidsand contaminate the liquid contained therein, is washed away.

Subsequently, both the washing liquid and the contaminated liquid areejected into a waste container. The nucleic acids are now immobilized inthe transfer container in a relatively pure form.

Then another protective tip is mounted, so that the lower part of thefirst protective tip is protected by the second protective tip. Thisprevents liquid which may still adhere to the first protective tip doesfrom coming into contact with the third liquid.

In another step, the transfer container is so immersed into the liquidthat the opening of the second tip extends into this liquid. The thirdliquid is one which causes the nucleic acid to disimmobilize from theglass fleece. This is in particular a liquid with a low salt buffer. Assoon as a sufficient amount of liquid was absorbed and the nucleic acidshave dissolved, this liquid together with the eluted nucleic acids arepoured, after a possible waiting period, into a new container. In thisnew container, the nucleic acids can be further treated, e.g. subject toa qualitative or quantitative determination. To accomplish this, thecontainer may already contain reagents in a solid or liquid form or suchreagents may be added later on.

To complete the method, the transfer container can be taken off thepipetting arm together with the protective tips and be discarded. Thisis preferably done by the pipetting instrument. In order to carry outanother determination a new transfer container is placed on theinstrument.

A significant advantage of the method of the invention is that it allowsthe combination of different tests in the manner described above,depending on whether a transfer container in accordance with theinvention or simple pipette tips are placed on the pipetting arm.

Whereas the above-described method proposes to remove the liquid fromthe lower opening of the transfer container and the protective tips, itis also conceivable that the liquids are discarded from the transfercontainer through the second opening. This possibility is used inparticular when the nucleic acids are immobilized to a solid phase andfor the washing steps. It is, however, also possible to draw in theeluted nucleic acids and carry out a measurement in a flow-throughmeter.

A protective tip can according to the invention be attached, if acontamination should be avoided from the transfer container or theprotective tip attached thereto into the (new) liquid container (in caseof transfer of a further amount of liquid from the already used liquidcontainer or in case of a transfer of an amount of (new) liquid from afurther liquid container). In this way it is for example possible toprevent that parts of already used washing liquid adherent to thetransfer container or to the pipette tip are transferred into the newwashing liquid and contaminate it. This contamination could yield inincorrect analyses results for analysis performed thereafter usingwashing liquid from the same washing liquid container.

FIG. 2 is a diagrammatic representation showing a pipetting instrument(01) with a pipetting arm (02), transfer containers (10) and (partly) ina magazine (12) protective tips (20), and in a magazine (22) reagents(41) (e.g. washing liquids and elution liquids in a reagent magazine(42)), and sample liquids in sample containers (52).

List of reference numerals

01 Pipetting instrument

02 Pipetting arm

10 Transfer container

11 Opening in 10

12 Magazine for 10

20 Protective tip

21 Opening in 20

22 Magazine for 20

30 Second protective tip

31 Opening in 30

41 Reagents

42 Magazine for 4 liters

52 Sample storage container

We claim:
 1. A transfer container in the form of a pipette tip andcontaining an agent for immobilizing a component of a sample liquid,said transfer container having a first opening therein at the tipthereof, and a protective tip having an opening therein mounted on thetip of the transfer container.
 2. A transfer container as recited inclaim 1, wherein said transfer container is a plastic pipette tip andsaid protective tip is a plastic pipette tip.
 3. A transfer container arecited in claim 1, wherein said protective tip makes a snap onconnection to the transfer container.
 4. A transfer container as recitedin claim 1, wherein said transfer container contains a glass fleece. 5.A transfer container as recited in claim 1, wherein said first openinghas a cross-sectional area of 0.15 mm².
 6. A transfer container asrecited in claim 1, wherein said transfer container has inside andoutside surfaces which are essentially conical.
 7. A transfer containeras recited in claim 1, wherein said agent for immobilizing a componentcomprises at least one material selected from the group consisting ofpolyester, polyamide, polycarbonate, cellulose, nitrocellulose andglass.
 8. A transfer container as recited in claim 1, wherein said agentfor immobilizing a component is fixed with an inert plastic net.
 9. Akit for the determination of an analyte in a sample liquid, comprisingat least one transfer container in the form of a pipette tip and havingan opening therein at the tip thereof, the transfer container containingan agent to immobilize the analyte in the transfer container, at leastone first protective tip mountable on the tip of the transfer containerand having a tip with a first protective tip opening therein, and atleast one second protective tip mountable on the tip of the firstprotective tip and having a tip with a second protective tip openingtherein.
 10. Kit of claim 9, wherein the first protective tip and thesecond protective tip are the same shape.
 11. Kit of claim 9, furtherincluding separately packaged washing liquid and elution liquid.
 12. Akit as recited in claim 9, wherein said at least one transfer containeris a plastic pipette tip, said at least one first protective tip is aplastic pipette tip, and said at least one second protective tip is aplastic pipette tip.
 13. A kit as recited in claim 9, wherein said atleast one first protective tip make a snap on connection to the at leastone transfer container.
 14. A kit as recited in claim 9, wherein said atleast one transfer container contains a glass fleece.
 15. A kit asrecited in claim 9, wherein said opening in said at least one transfercontainer has a cross-sectional area of 0.1-5 mm².
 16. A kit as recitedin claim 9, wherein said at least one transfer container has inside andoutside surfaces which are essentially conical.
 17. A kit as recited inclaim 9, wherein said agent to immobilize the analyte comprises at leastone material selected from the group consisting of polyester, polyamide,polycarbonate, cellulose, nitrocellulose and glass.
 18. A kit as recitedin claim 9, wherein said agent to immobilize the analyte is fixed withan inert plastic net.